Detailed protocol for Johnsen scoring in testicular histopathology

Detailed protocol for Johnsen scoring in testicular histopathology used in our study

Background

The Johnsen scoring system is a semiquantitative histopathological method used to evaluate spermatogenesis in seminiferous tubules. It assigns a score from 1 to 10 according to the most advanced germ cell type present and the degree of spermatogenic maturation. Higher scores indicate more advanced spermatogenesis, and a score of 8 or higher generally indicates the presence of spermatozoa.

Specimens

Testicular tissue specimens were obtained from patients with azoospermia who underwent testicular sperm extraction (TESE). A portion of the testicular tissue was submitted for pathological examination to assess intratesticular spermatogenesis.

Histological preparation

Testicular tissue was processed according to standard histopathological procedures, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histological evaluation was performed using light microscopy.

Microscopic evaluation

The histopathological findings of seminiferous tubules were assessed by an experienced pathologist. In our image-based study, microscopic images were collected at a fixed magnification of ×400 in order to maintain consistency in image acquisition and reduce bias related to image scale. This protocol reflects the practical histopathological approach used in our study.

Each seminiferous tubule cross-section was evaluated according to the most advanced germ cell type identified within the tubule, and a Johnsen score from 1 to 10 was assigned.

Johnsen scoring criteria

In general, scores of 8 to 10 indicate the presence of spermatozoa, whereas lower scores indicate maturation arrest or severe impairment of spermatogenesis.

Practical interpretation

For routine histopathological interpretation, each seminiferous tubule is scored individually. The overall spermatogenic status of the specimen is then interpreted based on the distribution of scores across representative tubules.

When there is heterogeneity among tubules, it is important to record the dominant pattern and note the presence of focal areas with more advanced or less advanced spermatogenesis.

Grouping used in our study

In our study, for practical and clinical purposes, the original 10-point Johnsen score was further grouped into four categories:

• Label 1 (scores 1–3): Differentiation up to spermatogonia
• Label 2 (scores 4–5): Differentiation up to spermatocytes
• Label 3 (scores 6–7): Differentiation up to spermatids
• Label 4 (scores 8–10): Differentiation up to spermatozoa

This simplified grouping was used because a 10-level classification was considered unnecessarily detailed for the clinical objective of our image classification study.

Notes and important points

1. Consistency of magnification is important. In our study, all images were collected at ×400 magnification. Mixing images obtained at different magnifications may reduce reproducibility.
2. Representative tubules should be selected carefully. Distorted, crushed, or poorly preserved tubules should be avoided when possible.
3. The score is based on the most advanced germ cell type present, but the general organization of spermatogenesis should also be considered.
4. Interobserver variation is possible, so evaluation by an experienced pathologist is recommended.
5. In specimens with markedly heterogeneous spermatogenesis, it is useful to describe both the predominant pattern and focal higher-grade areas.

Application in our study

Using this histopathological framework, we classified testicular histology images obtained from azoospermic patients who underwent TESE. In total, pathological specimens from 264 cases were analyzed, and histological images were grouped according to the four-category system described above. This grouping was adopted for practical use in our image-classification study rather than as a replacement for the original 10-point Johnsen system.

Conclusion

The Johnsen scoring system is a useful and widely accepted method for semiquantitative assessment of spermatogenesis in testicular tissue. It can be applied both in conventional pathology and in image-based analytical studies, provided that specimen handling, microscopy conditions, and scoring criteria are standardized.